4.2 Citric Acid Cycle

Overview & Subcellular Localization of the Citric Acid Cycle

The Citric Acid Cycle (also called the TCA Cycle or Krebs Cycle) is the central metabolic hub of eukaryotic aerobic organisms. Located within the mitochondrial matrix (with the exception of succinate dehydrogenase, which is bound to the inner mitochondrial membrane), the TCA cycle oxidizes two-carbon acetyl groups derived from carbohydrates, lipids, and amino acids into two molecules of $\text{CO}_2$.

The primary energetic function of the Citric Acid Cycle is not direct ATP production, but rather the harvesting of high-energy electrons. These electrons are captured by nucleotide coenzymes to form NADH and $\text{FADH}_2$, which subsequently feed into the electron transport chain to drive oxidative phosphorylation.

 Acetyl-CoA (2C) + Oxaloacetate (4C) ────────► Citrate (6C)
        ▲                                            │
        │                                            ▼ (TCA Cycle: 8 Steps)
 1 GTP + 1 FADH2 + 3 NADH ◄────────────────── 2 CO2 + Oxaloacetate (4C)

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The Eight Enzymatic Reactions of the TCA Cycle

Step 1: Condensation to Form Citrate

Citrate Synthase joins the two-carbon acetyl group of Acetyl-CoA with four-carbon Oxaloacetate (OAA) and water to form six-carbon Citrate and free $\text{CoA-SH}$:

$\text{Acetyl-CoA} + \text{Oxaloacetate} + \text{H}_2\text{O} \xrightarrow{\text{Citrate Synthase}} \text{Citrate} + \text{CoA-SH} + \text{H}^+ \quad (\Delta G^{\circ\prime} = -31.5\text{ kJ/mol})$

This reaction is highly exergonic and irreversible. The cleavage of the high-energy thioester bond of Acetyl-CoA pulls the reaction forward, keeping matrix OAA concentrations extremely low.

Step 2: Isomerization to Isocitrate

Aconitase (Aconitate Hydratase) reversibly isomerizes citrate to Isocitrate via a cis-aconitate intermediate ($\Delta G^{\circ\prime} = +6.3\text{ kJ/mol}$). Aconitase contains an essential iron-sulfur ($[4\text{Fe}-4\text{S}]$) cluster required for binding water and hydroxyl group transfer. Fluoroacetate poisons aconitase after conversion to fluorocitrate.

Step 3: First Oxidative Decarboxylation (Rate-Limiting Step)

Isocitrate Dehydrogenase oxidizes and decarboxylates six-carbon isocitrate to five-carbon $\alpha$-Ketoglutarate:

$\text{Isocitrate} + \text{NAD}^+ \xrightarrow{\text{Isocitrate Dehydrogenase, Mn}^{2+}} \alpha\text{-Ketoglutarate} + \text{NADH} + \text{CO}_2 \quad (\Delta G^{\circ\prime} = -20.9\text{ kJ/mol})$

This is the primary rate-limiting enzyme of the Citric Acid Cycle. It produces the first molecule of NADH and the first molecule of $\text{CO}_2$.

• Activated by: ADP (signals low energy) and $\text{Ca}^{2+}$.
• Inhibited by: ATP and NADH.

Step 4: Second Oxidative Decarboxylation

The $\alpha$-Ketoglutarate Dehydrogenase Complex converts five-carbon $\alpha$-ketoglutarate to four-carbon Succinyl-CoA:

$\alpha\text{-Ketoglutarate} + \text{CoA-SH} + \text{NAD}^+ \rightarrow \text{Succinyl-CoA} + \text{NADH} + \text{CO}_2 \quad (\Delta G^{\circ\prime} = -33.5\text{ kJ/mol})$

This irreversible step produces the second NADH and second $\text{CO}_2$. The enzyme complex is structurally and functionally homologous to the PDH complex, utilizing the exact same five cofactors (TPP, Lipoic Acid, CoA, FAD, $\text{NAD}^+$).

Step 5: Substrate-Level Phosphorylation

Succinyl-CoA Synthetase (Succinate Thiokinase) cleaves the high-energy thioester bond of Succinyl-CoA, coupling it to the phosphorylation of GDP (or ADP) to yield Succinate and GTP (or ATP):

$\text{Succinyl-CoA} + \text{GDP} + \text{P}_i \xrightleftharpoons{\text{Succinyl-CoA Synthetase}} \text{Succinate} + \text{GTP} + \text{CoA-SH} \quad (\Delta G^{\circ\prime} = -2.9\text{ kJ/mol})$

GTP is readily interconverted to ATP by Nucleoside Diphosphate Kinase ($\text{GTP} + \text{ADP} \rightleftharpoons \text{GDP} + \text{ATP}$).

Step 6: Oxidation of Succinate to Fumarate (Complex II)

Succinate Dehydrogenase oxidizes succinate to four-carbon Fumarate:

$\text{Succinate} + \text{FAD} \xrightleftharpoons{\text{Succinate Dehydrogenase}} \text{Fumarate} + \text{FADH}_2 \quad (\Delta G^{\circ\prime} = 0\text{ kJ/mol})$

Inner Membrane Bound (Complex II): Succinate Dehydrogenase is the only TCA cycle enzyme embedded in the inner mitochondrial membrane. It acts directly as Complex II of the Electron Transport Chain. It contains covalently bound $\text{FAD}$ and iron-sulfur centers. Malonate is a classic competitive inhibitor of succinate dehydrogenase due to its structural resemblance to succinate.

Step 7: Hydration of Fumarate to L-Malate

Fumarase (Fumarate Hydratase) catalyzes the stereospecific trans-addition of water across the double bond of fumarate to produce L-Malate ($\Delta G^{\circ\prime} = -3.8\text{ kJ/mol}$).

Step 8: Oxidation of L-Malate to Regeneration of Oxaloacetate

Malate Dehydrogenase oxidizes L-Malate to regenerate Oxaloacetate (OAA):

$\text{L-Malate} + \text{NAD}^+ \xrightleftharpoons{\text{Malate Dehydrogenase}} \text{Oxaloacetate} + \text{NADH} + \text{H}^+ \quad (\Delta G^{\circ\prime} = +29.7\text{ kJ/mol})$

Although this final reaction has a highly unfavorable standard free energy change ($\Delta G^{\circ\prime} = +29.7\text{ kJ/mol}$), it proceeds rapidly in vivo because Citrate Synthase (Step 1) continuously depletes oxaloacetate, keeping $[\text{OAA}]$ extremely low ($<10^{-6}\text{ M}$) and pulling the reaction forward.

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Summary Table: Reactions of the Citric Acid Cycle

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Energetics, Electron Capture & Cumulative Stoichiometry

Stoichiometry per Acetyl-CoA (Single Turn of TCA Cycle):

$\text{Acetyl-CoA} + 3\text{ NAD}^+ + \text{FAD} + \text{GDP} + \text{P}_i + 2\text{ H}_2\text{O} \rightarrow 2\text{ CO}_2 + 3\text{ NADH} + \text{FADH}_2 + \text{GTP} + \text{CoA-SH} + 3\text{ H}^+$

Cumulative Stoichiometry per 1 Molecule of D-Glucose:

One glucose molecule produces $2\text{ pyruvate}$, which yield $2\text{ Acetyl-CoA}$, driving two complete turns of the TCA cycle:

1. Glycolysis (Cytosol): $2\text{ ATP} + 2\text{ NADH}$
2. Pyruvate Dehydrogenase (Matrix): $2\text{ NADH} + 2\text{ CO}_2$
3. TCA Cycle (Matrix): $6\text{ NADH} + 2\text{ FADH}_2 + 2\text{ GTP} + 4\text{ CO}_2$
4. Total Cumulative Yield per Glucose: $4\text{ ATP/GTP} + 10\text{ NADH} + 2\text{ FADH}_2 + 6\text{ CO}_2$

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Amphibolic Nature & Anaplerotic Reactions

The Citric Acid Cycle is an amphibolic pathway, meaning it functions in both catabolic breakdown and anabolic precursor generation.

Cataplerotic Pathways (Draining Intermediates)

1. Citrate: Transported out of mitochondria into cytosol to yield Acetyl-CoA for fatty acid and cholesterol synthesis.
2. $\alpha$-Ketoglutarate: Transaminated to form Glutamate, leading to synthesis of purine nucleotides and other amino acids.
3. Succinyl-CoA: Condensed with glycine by $\delta$-aminolevulinic acid synthase (ALAS) to synthesize porphyrins and heme.
4. Oxaloacetate: Transaminated to Aspartate or converted to PEP for gluconeogenesis.

Anaplerotic Reactions (Replenishing Intermediates)

To prevent cycle shutdown when intermediates are removed for biosynthesis, anaplerotic reactions replenish TCA cycle pools:

• Pyruvate Carboxylase (Major Anaplerotic Step): Directs pyruvate to oxaloacetate inside the matrix: $\text{Pyruvate} + \text{CO}_2 + \text{ATP} + \text{H}_2\text{O} \xrightarrow{\text{Biotin, Acetyl-CoA}} \text{Oxaloacetate} + \text{ADP} + \text{P}_i$ This enzyme requires Biotin (Vitamin B7) and is obligately activated by Acetyl-CoA.
• Glutamate Dehydrogenase: Converts glutamate to $\alpha$-ketoglutarate.
• Odd-Chain Fatty Acid Oxidation: Yields propionyl-CoA, which is converted into succinyl-CoA.